The yeast rapid tRNA decay pathway primarily monitors the structural integrity of the acceptor and T-stems of mature tRNA

tRNAs, like other RNAs, are subject to quality control steps during and after biosynthesis. We previously described a rapid tRNA degradation (RTD) pathway in which the 5'-3' exonucleases Rat1 and Xrn1 degrade mature tRNA(Val(AAC)) in yeast mutants lacking m(7)G and m(5)C, and mature tRNA(Ser(CGA)) in mutants lacking Um and ac(4)C. To understand how the RTD pathway selects substrate tRNAs among different tRNAs lacking the same modifications, we used a genetic screen to examine tRNA(Ser(CGA)) variants. Our results suggest that RTD substrate recognition in vivo depends primarily on the stability of the acceptor and T-stems, and not the anti-codon stem, and does not necessarily depend on modifications, since fully modified tRNAs are subject to RTD if appropriately destabilized. We found that weaker predicted stability of the acceptor and T-stems of tRNAs is strongly correlated with RTD sensitivity, increased RNase T2 sensitivity of this region of the tRNA in vitro, and increased exposure of the 5' end to phosphatase. We also found that purified Xrn1 selectively degrades RTD substrate tRNAs in vitro under conditions in which nonsubstrates are immune. These results suggest that tRNAs have evolved not only for accurate translation, but for resistance to attack by RTD.